Compartment-driven imprinting of intestinal CD4 T cells in inflammatory bowel disease and homeostasis.

The mucosal immune system is implicated in the etiology and progression of inflammatory bowel diseases. The lamina propria and epithelium of the gut mucosa constitute two separate compartments, containing distinct T-cell populations. Human CD4 T-cell programming and regulation of lamina propria and epithelium CD4 T cells, especially during inflammation, remain incompletely understood. We performed flow cytometry, bulk, and single-cell RNA-sequencing to profile ileal lamina propria and intraepithelial CD4 T cells (CD4CD8αα, regulatory T cells (Tregs), CD69- and CD69high Trm T cells) in controls and Crohn's disease (CD) patients (paired non-inflamed and inflamed). Inflammation results in alterations of the CD4 T-cell population with a pronounced increase in Tregs and migrating/infiltrating cells. On a transcriptional level, inflammation within the epithelium induced T-cell activation, increased IFNγ responses, and an effector Treg profile. Conversely, few transcriptional changes within the lamina propria were observed. Key regulators including the chromatin remodelers ARID4B and SATB1 were found to drive compartment-specific transcriptional programming of CD4 T(reg) cells. In summary, inflammation in CD patients primarily induces changes within the epithelium and not the lamina propria. Additionally, there is compartment-specific CD4 T-cell imprinting, driven by shared regulators, between the lamina propria and the epithelium. The main consequence of intraepithelial adaptation, irrespective of inflammation, seems to be an overall dampening of broad (pro-inflammatory) responses and tight regulation of lifespan. These data suggest differential regulation of the lamina propria and epithelium, with a specific regulatory role in the inflamed epithelium.

Healthy Cotwins Share Gut Microbiome Signatures With Their Inflammatory Bowel Disease Twins and Unrelated Patients.

BACKGROUND & AIMS: It is currently unclear whether reported changes in the gut microbiome are cause or consequence of inflammatory bowel disease (IBD). Therefore, we studied the gut microbiome of IBD-discordant and -concordant twin pairs, which offers the unique opportunity to assess individuals at increased risk of developing IBD, namely healthy cotwins from IBD-discordant twin pairs. METHODS: Fecal samples were obtained from 99 twins (belonging to 51 twin pairs), 495 healthy age-, sex-, and body mass index-matched controls, and 99 unrelated patients with IBD. Whole-genome metagenomic shotgun sequencing was performed. Taxonomic and functional (pathways) composition was compared among healthy cotwins, IBD-twins, unrelated patients with IBD, and healthy controls with multivariable (ie, adjusted for potential confounding) generalized linear models. RESULTS: No significant differences were observed in the relative abundance of species and pathways between healthy cotwins and their IBD-twins (false discovery rate <0.10). Compared with healthy controls, 13, 19, and 18 species, and 78, 105, and 153 pathways were found to be differentially abundant in healthy cotwins, IBD-twins, and unrelated patients with IBD, respectively (false discovery rate <0.10). Of these, 8 (42.1%) of 19 and 1 (5.6%) of 18 species, and 37 (35.2%) of 105 and 30 (19.6%) of 153 pathways overlapped between healthy cotwins and IBD-twins, and healthy cotwins and unrelated patients with IBD, respectively. Many of the shared species and pathways have previously been associated with IBD. The shared pathways include potentially inflammation-related pathways, for example, an increase in propionate degradation and L-arginine degradation pathways. CONCLUSIONS: The gut microbiome of healthy cotwins from IBD-discordant twin pairs displays IBD-like signatures. These IBD-like microbiome signatures might precede the onset of IBD. However, longitudinal follow-up studies are needed to infer a causal relationship.

Right-Sided Location Not Associated With Missed Colorectal Adenomas in an Individual-Level Reanalysis of Tandem Colonoscopy Studies.

BACKGROUND & AIMS: Interval cancers occur more frequently in the right colon. One reason could be that right-sided adenomas are frequently missed in colonoscopy examinations. We reanalyzed data from tandem colonoscopies to assess adenoma miss rates in relation to location and other factors. METHODS: We pooled data from 8 randomized tandem trials comprising 2218 patients who had diagnostic or screening colonoscopies (adenomas detected in 49.8% of patients). We performed a mixed-effects logistic regression with patients as cluster effects with different independent parameters. Factors analyzed included location (left vs right, splenic flexure as cutoff), adenoma size, form, and histologic features. Analyses were controlled for potential confounding factors such as patient sex and age, colonoscopy indication, and bowel cleanliness. RESULTS: Right-side location was not an independent risk factor for missed adenomas (odds ratio [OR] compared with the left side, 0.94; 95% CI, 0.75-1.17). However, compared with adenomas ≤5 mm, the OR for missing adenomas of 6-9 mm was 0.62 (95% CI, 0.44-0.87), and the OR for missing adenomas of ≥10 mm was 0.51 (95% CI, 0.33-0.77). Compared with pedunculated adenomas, sessile (OR, 1.82; 95% CI, 1.16-2.85) and flat adenomas (OR, 2.47; 95% CI, 1.49-4.10) were more likely to be missed. Histologic features were not significant risk factors for missed adenomas (OR for adenomas with high-grade intraepithelial neoplasia, 0.68; 95% CI, 0.34-1.37 and OR for sessile serrated adenomas, 0.87; 95% CI, 0.47-1.64 compared with low-grade adenomas). Men had a higher number of adenomas per colonoscopy (1.27; 95% CI, 1.21-1.33) than women (0.86; 95% CI, 0.80-0.93). Men were less likely to have missed adenomas than women (OR for missed adenomas in men, 0.73; 95% CI, 0.57-0.94). CONCLUSIONS: In an analysis of data from 8 randomized trials, we found that right-side location of an adenoma does not increase its odds for being missed during colonoscopy but that adenoma size and histologic features do increase risk. Further studies are needed to determine why adenomas are more frequently missed during colonoscopies in women than men.

Systematic Review and External Validation of Prediction Models Based on Symptoms and Biomarkers for Identifying Endoscopic Activity in Crohn's Disease.

BACKGROUND & AIMS: Endoscopic healing, an important target of treatment for Crohn's disease (CD), requires ileocolonoscopy, which is costly and burdensome. We investigated whether published noninvasive models (based on symptoms and biomarkers) to evaluate CD activity have sufficient accuracy to replace ileocolonoscopy. METHODS: We performed a systematic review of published noninvasive diagnostic models to evaluate CD activity that used endoscopic features of activity (endoscopic activity) or healing as the reference standard. We externally validated these models for the outcome endoscopic activity (CD endoscopic index of severity scores, ≥3) using data from the a randomized controlled trial investigating tailored treatment with infliximab for active luminal Crohn's disease (TAILORIX) study (346 ileocolonoscopies in 155 patients) and the Utrecht Activity Index (UAI) study (93 ileocolonoscopies in 82 patients). We calculated the area under the receiver operating characteristic curves (AUROCs) for the models using data from these studies, and compared the performance of these models against measurements of fecal calprotectin (FC) and C-reactive protein (CRP). RESULTS: We screened 5303 articles and identified 27 models (from 21 studies) for our analysis. Seven models could be validated externally; in the TAILORIX data set, these models identified patients with endoscopic activity with AUROC values ranging from 0.61 (95% CI, 0.51-0.70) to 0.81 (95% CI, 0.76-0.86). In this data set, the AUROC value for FC concentration was 0.79 (95% CI, 0.74-0.85) and the AUROC value for CRP level was 0.72 (95% CI, 0.66-0.77). The AUROC values for the validation in the UAI data set were similar. In the TAILORIX and/or UAI data set, 4 of the 7 models, as well as the FC and CRP assays, were able to identify patients with endoscopic activity with positive predictive values of 90% or more. Two of the 7 models (but not the FC or CRP values) identified patients without endoscopic activity with a negative predictive value (NPV) of 90% or more, leading to correct prediction of endoscopic healing in 3.2% to 11.3% of all patients. For example, applying the Herranz-Bachiller model (1 of 7 models) at a NPV of 92.1% and a positive predictive value of 91.9% correctly identified 35.7% of all patients in whom ileocolonoscopy could be avoided for expected endoscopic activity or healing but incorrectly identified 3.2% of all patients. Most ileocolonoscopies (66.5% in TAILORIX and 72.6% in the UAI of all ileocolonoscopies) could be avoided correctly based on concentrations of FC of 100 µg/g or less and 250 µg/g or higher. However, using this range of FC concentrations to identify patients who do not require ileocolonoscopy caused 18.7% of all patients in the TAILORIX cohort and 19.8% of all patients in the UAI cohort to be predicted incorrectly to have endoscopic activity or healing. CONCLUSIONS: In a systematic review and external validation of noninvasive models to identify patients with endoscopic activity of CD, we found only 2 of 7 models evaluated to have NPVs of 90% or more, however, leading to correctly predicted EH in only a small proportion of patients. Ileocolonoscopy therefore remains the mainstay to evaluate CD mucosal disease activity and healing.

A systematic review and meta-analysis of complication rates after carotid procedures performed by different specialties.

OBJECTIVE: Different competencies and skills are required and obtained during medical specialization. However, whether these have an impact on procedural outcomes of carotid endarterectomy (CEA) or carotid artery stenting (CAS) is unclear. We assessed the reported association between operator specialization and procedural outcomes after CEA or CAS to determine whether CEA and CAS should be performed by specific specialties. METHODS: We systematically searched PubMed and Embase up to August 21, 2017, for randomized clinical trials and observational studies that compared two or more specialties performing CEA or CAS for symptomatic and asymptomatic carotid artery stenosis. The composite primary outcome was procedural stroke or death (ie, occurring within 30 days of the procedure or before discharge). Risk estimates were pooled with a generic inverse variance random effects model. RESULTS: A total of 35 studies (26 providing data on CEA, 8 providing data on CAS, and 1 providing data on both CEA and CAS) were included, describing 256,033 CEA and 38,605 CAS procedures. For CEA, decreased risk of procedural stroke or death for operations performed by vascular surgeons was found with pooled unadjusted relative risk (RR) of 0.63 (95% confidence interval [CI], 0.46-0.86; seven studies) compared with neurosurgeons and RR of 0.81 (95% CI, 0.66-0.99; six studies) compared with general surgeons. An increased risk of procedural stroke or death for operations performed by neurosurgeons compared with cardiothoracic surgeons was found with a pooled unadjusted RR of 1.22 (95% CI, 1.02-1.46). No studies adjusted for potential confounding, and no significant unadjusted associations were found in other comparisons of operator specialty for the primary outcome. For CAS, no differences in procedural stroke or death were found by operator specialty. CONCLUSIONS: Studies were at high risk of bias mainly because of potential confounding by patient selection for CEA and CAS. Current evidence is insufficient to restrict CEA or CAS to specific specialties.

Anti-tumor necrosis factor therapy in patients with inflammatory bowel disease; comorbidity, not patient age, is a predictor of severe adverse events.

PURPOSE: To assess safety and effectiveness of anti-tumor necrosis factor (anti-TNF) therapy in IBD patients ≥ 60 years. METHODS: Ninety IBD patients ≥ 60 years at initiation of anti-TNF therapy, 145 IBD patients ≥ 60 years without anti-TNF therapy and 257 IBD patients < 60 years at initiation of anti-TNF therapy were retrospectively included in this multicentre study. Primary outcome was the occurrence of severe adverse events (SAEs), serious infections and malignancies. Secondary outcome was effectiveness of therapy. Cox regression analyses were used to assess differences in safety and effectiveness. In safety analyses, first older patients with and without anti-TNF therapy and then older and younger patients with anti-TNF therapy were assessed. RESULTS: In older IBD patients, the use of anti-TNF therapy was associated with serious infections (aHR 3.920, 95% CI 1.185-12.973, p = .025). In anti-TNF-exposed patients, cardiovascular disease associated with serious infections (aHR 3.279, 95% CI 1.098-9.790, p = .033) and the presence of multiple comorbidities (aHR 9.138 (1.248-66.935), p = .029) with malignancies, while patient age did not associate with safety outcomes. Effectiveness of therapy was not affected by age or comorbidity. CONCLUSION: Older patients receiving anti-TNF therapy have a higher risk of serious infections compared with older IBD patients without anti-TNF therapy, but not compared with younger patients receiving anti-TNF therapy. However, in anti-TNF-exposed patients, comorbidity was found to be an indicator with regards to SAEs. Effectiveness was comparable between older and younger patients.

High Operator and Hospital Volume Are Associated With a Decreased Risk of Death and Stroke After Carotid Revascularization: A Systematic Review and Meta-analysis.

OBJECTIVE: To examine the association between operator or hospital volume and procedural outcomes of carotid revascularization. BACKGROUND: Operator and hospital volume have been proposed as determinants of outcome after carotid endarterectomy (CEA) or carotid artery stenting (CAS). The magnitude and clinical relevance of this relationship are debated. METHODS: We systematically searched PubMed and EMBASE until August 21, 2017. The primary outcome was procedural (30 days, in-hospital, or perioperative) death or stroke. Obtained or estimated risk estimates were pooled with a generic inverse variance random-effects model. RESULTS: We included 87 studies. A decreased risk of death or stroke following CEA was found for high compared to low operator volume with a pooled adjusted odds ratio (OR) of 0.50 (95% confidence interval [CI] 0.28-0.87; 3 cohorts), and a pooled unadjusted relative risk (RR) of 0.59 (95% CI 0.42-0.83; 9 cohorts); for high compared to low hospital volume with a pooled adjusted OR of 0.62 (95% CI 0.42-0.90; 5 cohorts), and a pooled unadjusted RR of 0.68 (95% CI 0.51-0.92; 9 cohorts). A decreased risk of death or stroke after CAS was found for high compared to low operator volume with an adjusted OR of 0.43 (95% CI 0.20-0.95; 1 cohort), and an unadjusted RR of 0.50 (95% CI 0.32-0.79; 1 cohort); for high compared to low hospital volume with an adjusted OR of 0.46 (95% CI 0.26-0.80; 1 cohort), and no significant decreased risk in a pooled unadjusted RR of 0.72 (95% CI 0.49-1.06; 2 cohorts). CONCLUSIONS: We found a decreased risk of procedural death and stroke after CEA and CAS for high operator and high hospital volume, indicating that aiming for a high volume may help to reduce procedural complications. REGISTRATION: This systematic review has been registered in the international prospective registry of systematic reviews (PROSPERO): CRD42017051491.

Plasma fibrinogen level as a potential predictor of hemorrhagic complications after catheter-directed thrombolysis for peripheral arterial occlusions.

BACKGROUND: The benefit of catheter-directed thrombolysis for peripheral arterial occlusions is limited by hemorrhagic complications. Plasma fibrinogen level (PFL) has been suggested as a predictor of these hemorrhagic complications, but the accurateness of prediction is unknown. We summarized the available evidence on the predictive value of PFL for hemorrhagic complications after catheter-directed thrombolysis for acute or subacute peripheral native artery or arterial bypass occlusions. METHODS: We systematically searched PubMed and Embase until January 2016 for peer-reviewed publications on adults undergoing thrombolysis for acute or subacute peripheral native artery or arterial bypass occlusions, assessing the predictive value of PFL for hemorrhagic complications. Two authors independently performed data extraction. Risk of bias was assessed with the Quality in Prognosis Studies (QUIPS) tool. RESULTS: In total, six studies (two randomized clinical trials and four cohort studies) reported on 613 patients undergoing 623 thrombolytic interventions for peripheral native artery or arterial bypass occlusions. No risk estimates for PFL and hemorrhagic complications were reported, two risk estimates were calculated, and nine associations between PFL and hemorrhagic complications were reported. For PFL <100 mg/dL compared with ≥100 mg/dL, the calculated relative risk was 0.33 (95% confidence interval, 0.05-2.25) for major bleeding and 1.39 (95% confidence interval, 1.06-1.81) for any bleeding. There were considerable differences in the time point of PFL measurement, the thrombolytic agents, the doses of the agents, and the definition of outcomes. PFL seems inaccurate in predicting hemorrhagic complications. Overall, the included studies were at high risk of bias. CONCLUSIONS: Based on the current literature, the predictive value of PFL for predicting hemorrhagic complications after catheter-directed thrombolysis for acute or subacute peripheral native artery and arterial bypass occlusions is unproven.

Missed adenomas with behind-folds visualizing colonoscopy technologies compared with standard colonoscopy: a pooled analysis of 3 randomized back-to-back tandem colonoscopy studies.

BACKGROUND AND AIMS: The Third Eye Retroscope, Full Spectrum Endoscope (FUSE), and EndoRings devices have been shown to reduce overall adenoma miss rates. We evaluated the characteristics of adenomas and patient subgroups for which these behind-folds visualizing technologies mostly reduce adenoma miss rates. METHODS: Data of 3 multicenter randomized trials (NCT01044732, NCT01549535, NCT01955122) were combined. Patients underwent same-day, back-to-back tandem examinations with standard colonoscopy and Third Eye Retroscope, FUSE, or EndoRings colonoscopy, respectively. Adenoma miss rates were stratified by adenoma characteristics and patient subgroups. RESULTS: A total of 650 patients (60% male, mean age 57.5 years, standard deviation 9.7 years) were included; 330 patients underwent behind-folds visualizing colonoscopy first, and 320 patients underwent standard colonoscopy first. Regarding adenoma characteristics, adenoma miss rates were significantly (P < .001) lower with behind-folds visualizing technologies compared with standard colonoscopy for proximal (14% vs 38%) and distal (15% vs 35%), ≤5 mm (17% vs 38%), 6 to 9 mm (8% vs 44%), sessile (16% vs 37%), flat (9% vs 52%; P = .014), and tubular (15% vs 38%) adenomas and sessile serrated polyps (7% vs 50%; P = .039) but were not statistically significantly (P > .05) different for ≥10 mm, pedunculated, (tubulo-)villous, and advanced adenomas. Regarding patient subgroups, adenoma miss rates were significantly (P ≤ .020) lower with behind-folds visualizing technologies for patients ≥50 years, both sexes, and all indications. CONCLUSIONS: Behind-folds visualizing colonoscopy reduces miss rates for 1 to 9 mm adenomas in the entire colon, whereas no advantage was found for ≥10 mm and advanced adenomas. Whether increased detection and removal of <10 mm adenomas also reduces colorectal cancer incidence and mortality remains to be determined. Future research is needed to determine which colonoscopy technology would be most beneficial for which patient or endoscopist.

Posterior transverse plication of the internal carotid artery to correct for kinking.

BACKGROUND: The occasional need for shortening of the internal carotid artery (ICA) following carotid endarterectomy (CEA) to correct for kinking is still controversial. Although several technical options have been suggested, the impact on perioperative outcome remains unclear, and long-term clinical follow-up is lacking. Shortening by resection has a theoretical risk for a twisted anastomosis and subsequent ICA thrombosis. Posterior transverse plication (PTP) offers an alternative shortening technique without the need for a new anastomosis. We aimed to assess the safety and patency of CEA with concomitant PTP. Secondly, we aimed to provide an overview of different technical modalities for shortening of the carotid artery in current literature. METHODS: Within the time frame of 2000 through 2011, 29 patients (mean age, 73.4 years) undergoing CEA with additional PTP of the ICA and standardized patchplasty were retrospectively identified. Patient characteristics, surgical procedural details, and both short- (<30 days) and long- (>30 days) term clinical and duplex ultrasound follow-up were retrieved. Restenosis was defined as ≥50% stenosis on duplex ultrasound. In addition, a literature search was performed on different techniques for ICA shortening. RESULTS: Thirty-day outcome revealed no deaths or strokes. No postprocedural thrombosis or narrowing of the ipsilateral ICA was observed. During follow-up (mean, 34.3 months; range, 3-125 months), one patient (4%) died of a noncardiovascular cause. Three patients (11%) developed ipsilateral neurological symptoms (1 stroke, 2 transient ischemic attacks) after 5, 19, and 66 months follow-up, respectively. Of these, two patients (7%) had restenosis at the site of PTP. Asymptomatic restenosis occurred in one other patient (4%) after 16 months. CONCLUSIONS: Although the indications for additional shortening procedures following CEA need to be defined, in this small series, PTP as an additional shortening procedure of the ICA following CEA seems feasible and safe with no additional periprocedural risk for narrowing at the plicature or thrombosis of the endarterectomy plane. However, restenosis at the plicature may hamper the long term benefit of carotid reconstruction.

Next-generation IgA-SEQ allows for high-throughput, anaerobic, and metagenomic assessment of IgA-coated bacteria.

BACKGROUND: The intestinal microbiota plays a significant role in maintaining systemic and intestinal homeostasis, but can also influence diseases such as inflammatory bowel disease (IBD) and cancer. Certain bacterial species within the intestinal tract can chronically activate the immune system, leading to low-grade intestinal inflammation. As a result, plasma cells produce high levels of secretory antigen-specific immunoglobulin A (IgA), which coats the immunostimulatory bacteria. This IgA immune response against intestinal bacteria may be associated with the maintenance of homeostasis and health, as well as disease. Unraveling this dichotomy and identifying the immunostimulatory bacteria is crucial for understanding the relationship between the intestinal microbiota and the immune system, and their role in health and disease. IgA-SEQ technology has successfully identified immunostimulatory, IgA-coated bacteria from fecal material. However, the original technology is time-consuming and has limited downstream applications. In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol. RESULTS: We show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated bacteria in a high-throughput and time efficient manner. Furthermore, we performed a comparative analysis between different IgA-SEQ protocols, highlighting that the original FACS-based IgA-SEQ approach overlooks certain nuances of IgA-coated bacteria, due to the low yield of sorted bacteria. Additionally, magnetic-based ng-IgA-SEQ allows for novel downstream applications. Firstly, as a proof-of-concept, we performed metagenomic shotgun sequencing on 10 human fecal samples to identify IgA-coated bacterial strains and associated pathways and CAZymes. Secondly, we successfully isolated and cultured IgA-coated bacteria by performing the isolation protocol under anaerobic conditions. CONCLUSIONS: Our magnetic 96-well plate-based high-throughput next-generation IgA-SEQ technology efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for analyzing large cohorts as well as novel downstream applications, including shotgun metagenomic sequencing, culturomics, and various functional assays. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease. Video Abstract.

Highly multiplexed spatial analysis identifies tissue-resident memory T cells as drivers of ulcerative and immune checkpoint inhibitor colitis.

Colitis is a prevalent adverse event associated with immune checkpoint inhibitor (ICI) therapy with similarities to inflammatory bowel disease. Incomplete mechanistic understanding of ICI colitis curtails evidence-based treatment. Given the often-overlooked connection between tissue architecture and mucosal immune cell function, we here applied imaging mass cytometry (IMC) to gain spatial proteomic insight in ICI colitis in comparison to ulcerative colitis (UC). Using a cell segmentation pipeline that simultaneously utilizes high-resolution nuclear imaging and high-multiplexity IMC, we show that intra-epithelial CD8+ T cells are significantly more abundant (and numerically dominant) in anti-PD-1 ± anti-CTLA-4-induced colitis compared to anti-CTLA-4-induced colitis and UC. We identified activated, cycling CD8+ tissue-resident memory T(RM) cells at the lamina propria-epithelial interface as drivers of cytotoxicity in ICI colitis and UC. Moreover, we found that combined ICI-induced colitis featured highest granzyme B levels both in tissue and serum. Together, these data reinforce CD8+ TRM cells as potentially targetable drivers of ICI colitis.

[Antibiotic-induced acute kidney injury]. / Antibiotica-geïnduceerde acute nierinsufficiëntie.

Acute kidney injury is very common in hospitalized patients and has been described in up to twenty percent of admissions. Although there are many causes of acute kidney injury, one of the more overlooked causes is the antibiotic prescribed during these admissions. In this article we discuss the two main causes of antibiotic induced kidney injury illustrated by two cases, one of ciprofloxacin Kristal nephropathy and of ciprofloxacin tubule-interstial nephritis. We discuss the pathophysiology, most common involved antibiotics, diagnostics and the treatment.

Human regulatory T cells locally differentiate and are functionally heterogeneous within the inflamed arthritic joint.

Objective: Tregs are crucial for immune regulation, and environment-driven adaptation of effector (e)Tregs is essential for local functioning. However, the extent of human Treg heterogeneity in inflammatory settings is unclear. Methods: We combined single-cell RNA- and TCR-sequencing on Tregs derived from three to six patients with juvenile idiopathic arthritis (JIA) to investigate the functional heterogeneity of human synovial fluid (SF)-derived Tregs from inflamed joints. Confirmation and suppressive function of the identified Treg clusters was assessed by flow cytometry. Results: Four Treg clusters were identified; incoming, activated eTregs with either a dominant suppressive or cytotoxic profile, and GPR56+CD161+CXCL13+ Tregs. Pseudotime analysis showed differentiation towards either classical eTreg profiles or GPR56+CD161+CXCL13+ Tregs supported by TCR data. Despite its most differentiated phenotype, GPR56+CD161+CXCL13+ Tregs were shown to be suppressive. Furthermore, BATF was identified as an overarching eTreg regulator, with the novel Treg-associated regulon BHLHE40 driving differentiation towards GPR56+CD161+CXCL13+ Tregs, and JAZF1 towards classical eTregs. Conclusion: Our study reveals a heterogeneous population of Tregs at the site of inflammation in JIA. SF Treg differentiate to a classical eTreg profile with a more dominant suppressive or cytotoxic profile that share a similar TCR repertoire, or towards GPR56+CD161+CXCL13+ Tregs with a more distinct TCR repertoire. Genes characterising GPR56+CD161+CXCL13+ Tregs were also mirrored in other T-cell subsets in both the tumor and the autoimmune setting. Finally, the identified key regulators driving SF Treg adaptation may be interesting targets for autoimmunity or tumor interventions.

Homeostatic Function and Inflammatory Activation of Ileal CD8+ Tissue-Resident T Cells Is Dependent on Mucosal Location.

BACKGROUND & AIMS: Tissue-resident memory T (Trm) cells, both of the CD4 and CD8 lineage, have been implicated in disease flares in inflammatory bowel disease. However, data are conflicting regarding the profile of human CD8+ Trm cells, with studies suggesting both proinflammatory and regulatory functions. It is crucial to understand the functional profile of these cells in the context of (new) therapeutic strategies targeting (trafficking of) gut Trm cells. METHODS: Here, we performed imaging mass cytometry, flow cytometry, and RNA-sequencing to compare lamina propria and intraepithelial CD103+/-CD69+CD8+ Trm cells in healthy control subjects and patients with active ileal Crohn's disease. RESULTS: Our data revealed that lamina propria CD103+CD69+CD8+ T cells have a classical Trm cell profile with active pathways for regulating cell survival/death and cytokine signaling, whereas intraepithelial CD103+CD69+CD8+ T cells display tightly regulated innate-like cytotoxic profile. Furthermore, within lamina propria CD8+CD103- Trm cells, an Itgb2+GzmK+KLRG1+ population distinct from CD103+ CD8+ Trm cells is found. During chronic inflammation, especially intraepithelial CD103+CD69+CD8+ T cells displayed an innate proinflammatory profile with concurrent loss of homeostatic functions. CONCLUSIONS: Altogether, these compartmental and inflammation-induced differences indicate that therapeutic strategies could have a different impact on the same immune cells depending on the local compartment and presence of an inflammatory milieu, and should be taken into account when investigating short- and long-term effects of new gut T cell-targeting drugs.

Development of Mucosal PNAd+ and MAdCAM-1+ Venules during Disease Course in Ulcerative Colitis.

PNAd and MAdCAM-1 addressins on venules are of importance in T-cell homing and potential therapeutic targets in ulcerative colitis (UC). Normally, PNAd+ high endothelial venules (HEVs) are only present in lymphoid organs, whereas small numbers of MAdCAM-1+ venules can be seen in non-lymphoid tissue. We aimed to study their presence in the intestinal mucosa of UC patients at diagnosis and during follow-up, and their correlation with disease activity. Colonic biopsy specimens of 378 UC patients were analyzed by immunohistochemistry for CD3, CD20, ERG, MECA-79 (PNAd) and MECA-376 (MAdCAM-1) and compared to healthy controls (HC). The proportion of PNAd+HEVs in UC at diagnosis was 4.9% (IQR 2.0%-8.3%), while none were detected in HC. During follow-up, PNAd+HEVs completely disappeared in remission (n = 93), whereas the proportion in active disease was similar to baseline (n = 285, p = 0.39). The proportion of MAdCAM-1+venules in UC at baseline was 5.8% (IQR 2.6-10.0). During follow-up, the proportion in remission was comparable to diagnosis, but upregulated (7.5% (IQR 4.4-10.9), p = 0.001) in active disease. In conclusion, PNAd+HEVs appear in UC during active inflammation which could thus serve as a marker for disease activity, whereas MAdCAM-1+venules remain present after inflammation is resolved and increase after subsequent flares, reflecting chronicity and potentially serving as a therapeutic target.